Method and composition for prevention of scar formation in glaucoma filtration bleb and drainage fistula

ABSTRACT

This invention is related to a method whereby scar formation in the drainage fistula and subconjunctival bleb created during and after glaucoma surgery using a prostaglandin subtype A or J.

This is a U.S. National Phase Application Under 35 USC 371 and applicantherewith claims the benefit of priority of PCT/SE00/00357 filed Feb. 23,2000, which was published under PCT Article 21(2) in English andApplication No. 9900672-8 filed in Sweden on Feb. 25, 1999.

The present invention is related to a method whereby scar formation inthe drainage fistula and subconjunctival bleb created during glaucomasurgery can be prevented. The invention is also related to a compositionused for prevention of scar formation in the drainage fistula andsubconjunctival bleb after glaucoma surgery.

BACKGROUND OF THE INVENTION

Glaucoma is an eye disorder characterized by increased intraocularpressure, excavation of the optic nerve head, and gradual loss of visualfield. An abnormally high intraocular pressure is commonly known to bedetrimental to the eye, and there are clear indications that in glaucomathe intraocular pressure is the most important factor causingdegenerative changes in the retina and the optic nerve head. The exactpathophysiological mechanism of open angle glaucoma is, however, stillunknowns. Unless treated glaucoma may lead to blindness, the course ofthe disease typically being slow with progressive loss of vision.

The intraocular pressure is determined by the rate of production anddrainage of aqueous humor in the anterior part of the eye. The aqueoushumor is produced by the ciliary processes behind the iris. It thenflows through the pupil into the anterior chamber, and normally exitsthe eye through the trabecular meshwork and Schlemm's canal. However, inopen angle glaucoma the resistance to outflow of aqueous humor isincreased which causes the intraocular pressure to raise. Theintraocular pressure in humans is normally around 12-21 mmHg. At higherpressures there is an increased risk that the eye may be damaged. In oneparticular form of glaucoma, namely low tension glaucoma, damage may,however, occur at intraocular pressures regarded to be within the normalphysiological range. The opposite situation is also known i.e. someindividuals may exhibit abnormally high intraocular pressure without anymanifest defects in the visual field or optic nerve head. Suchconditions usually are referred to as ocular hypertension.

Glaucoma treatment can be given by means of drugs, laser or surgery.Usually surgery is employed only when drug and laser treatments nolonger are sufficiently effective. A relatively new medical treatment ofglaucoma comprises the use of prostaglandins, which are administeredtopically on the eye, and reduce the intraocular pressure by enhancingthe outflow of aqueous humor. Two such prostaglandin-based glaucomadrugs are currently being marketed in many countries, i.e. latanoprost(Xalatan®) and isopropyl unoprostone (Rescula®), and are extensivelyused clinically. The use of prostaglandins and derivatives is describedin several patents and patent applications e.g. U.S. Pat No. 4,599,353(Bito), U.S. Pat No. 4,952,581 (Bito), WO89/03384 (Sternschantz andResul), WO 96/09055 (Sternschantz and Resul), EP 170258 (Cooper), EP253094 (Goh) and EP 308135 (Ueno). In addition to these patents andpatent applications a large number of new patent applications have beenfiled during the last years. Common for all these patents and patentapplications is that they describe the use of prostaglandins forreduction of the intraocular pressure without surgery.

The prostaglandins are fatty acids usually derived from the precursorseicosatrienoic, eicosatetracnoic and eicosapentaenoic acid throughmetabolic steps involving oxygenation. Naturally occuring prostaglandinstypically have the general structure presented below:

The prostaglandins accordingly carry a cyclopentane ring to which twocarbon chains attach, the upper chain usually being called the alphachain and the lower chain usually being called the omega chain. Theprostaglandins are classified in subgroups A, B, C, D, E, F, G, H, I,and J, depending on the structure and the substituents in thecyclopentane ring. The prostaglandins of particular interest in thepresent invention belong to the subgroups A and J, and theircyclopentane ring configuration is presented below:

The alpha chain is a 7 carbon carboxy-terminated aliphatic chain whereasthe omega chain is an 8 carbon methyl-terminated aliphatic chain.Depending on the number of double bonds in these chains, subscripts of 1to 3 are given. In prostaglandins with subscript 1, e.g. PGA₁, thedouble bond is situated between carbons 13 and 14 in the omega chain. Inprostaglandins with subscript 2,e.g. PGJ₂, an additional double bond issituated between carbons 5 and 6 in the alpha chain, and finally inprostaglandins with subscript 3, a third double bond is situated betweencarbons 17 and 18 in the omega chain. All naturally occuringprostaglandins furthermore carry a hydroxyl group on carbon 15.

Many different techniques have been described for glaucoma surgery.However, all techniques aim at creating a small drainage fistula for theaqueous humor to exit the eye in the vicinity of the trabecularmeshwork. Thus the aqueous humor can bypass the trabecular meshworktissue at Schlemm's canal that usually is clogged in open angleglaucoma. The fluid is directed into a filtration bleb beneath theconjunctiva outside the eye. The most commonly practiced operationtechnique is called trabeculectomy and usually results in satisfactorypressure lowering of the eye. A very common complication, however, isformation of scar tissue in the filtration bleb, which reduces thedrainage capacity of the filtration system created by surgery. The scarformation is mainly due to the proliferation and increased activity offibroblasts. Consequently the intraocular pressure with time starts toreturn to pathological levels. Usually the scarring occurs severalmonths Lo yews after surgery, and the use of antimitotic agents such as5-fluorouracil and Mitomycin C during surgery improves the surgicalresults. However, Mitomycin C and 5-fluorouracil are very toxiccompounds with a narrow therapeutic index, and difficult to useclinically. Late complications such as conjunctival holes may ensueafter the use of e.g. Mitomycin C. Thus there is a need for better andsafer drugs to be used as a complement to the surgical procedure toprevent the scarring of the filtration system created by surgery.

SUMMARY OF THE INVENTION

The above problems associated with glaucoma surgery are now solved bythe present invention as defined in the attached claims, herebyincorporated by reference. The invention is based on the unexpectedfinding that prostaglandins of type A and J may be highly effective inpreventing scar formation that typically occurs after glaucoma surgery,and that the compositions comprising these compounds and the methodsdescribed have advantages over the compositions and methods hithertoknown.

SHORT DESCRIPTION OF THE DRAWINGS

The present invention is described below with reference to the attacheddrawings, in which

FIG. 1 shows the chemical structures of the tested compounds,prostaglandin A₁, prostaglandin A₂, prostaglandin J₂ (PG J₂) and15-deoxy-Δ^(12,14)-prostaglandin J₂ (^(15Δ)PGJ₂);

FIG. 2 shows the absorbance at 595 nm (measure of cell number) as afunction of the concentration (plotted as negative logarithm)of the testcompounds PGA₁, and PGA₂,

FIG. 3 shows the absorbance at 595 nm (measure of cell number) as afunction of the concentration (plotted as negative logarithm) of thetest compounds PGJ₂ and 15-deoxy-Δ^(12,14)-prostaglandin J₂(^(15Δ)PGJ₂),

FIG. 4 shows the intraocular pressure in the vehicle-treated operated,and the control eyes of rabbits following trabebeculectomy surgeryduring four months.

FIG. 5 shows the intraocular pressure in PGA₂-IE-treated operated, andthe control eyes of rabbits following trabebeculectomy surge during fourmonths.

DESCRIPTION

Experiments performed by the present inventors have shown that specificprostaglandins have a marked antimitotic effect on human fibroblasts inculture. Indeed, at high concentrations these prostaglandins seem toexert even cytotoxic effects, killing fibroblasts. Accordingly, topicaltreatment of the eye with such prostaglandins after surgery will preventscarring of the filtration bleb and other parts of the filtrationsystem, and thus help keeping the intraoclar pressure at a desiredreduced level.

According to one embodiment of the invention, prostaglandins of the Aand J type are used for the treatment of glaucoma after filtrationsurgery, particularly trabeculectomy, by preventing the proliferation offibroblasts in the filtration system. The method comprises contactingthe surface of the eye, including the filtration bleb, with an effectiveamount of a composition containing a prostaglandin of the subtype A orJ. The composition usually contains 0.1-500 μg, especially 1-30 μg perapplication of the active substance. The composition is appliedtopically on the eye 1-3 times daily, or only every second day, or oncea week, or possibly intermittently, e.g. for a month and thereafter afew months later again for a month etc.

The prostaglandin derivative is mixed with an ophthalmologicallycompatible vehicle known per se. Vehicles, which may be employed forpreparing compositions according to the present invention comprisesaqueous solutions, e.g. physiological saline, oil solutions, creams andointments. The vehicle may furthermore contain ophthalmologicallycompatible preservatives such as benzalkonium chloride, inclusioncomplexes such as cyclodextins, surfactants e.g. polysorbate 80, andliposomes. Polymeres, for example methyl cellulose, polyvinyl alcohol,polyvinyl pyrrolidone, and hyaluronicacid may also be employed; thesemay be used for increasing the viscosity. Furthermore it is alsopossible to use soluble or insoluble drug inserts.

One embodiment of the invention comprises ophthalmological compositions,for treatment of glaucoma after trabeculectomy to prevent scaring of thefiltration system, which comprise an effective amount of a prostaglandinof the A or J type, and an ophtalmologically compatible carrier. Theeffective amount usually comprises a dose of about 0.1-500 μg in about10-50 μl of the composition.

According to another embodiment, the invention relates to the use of aprostaglandin analogue for the preparation of a medicament for thetreatment of glaucoma after fistulating surgery to prevent scarformation in the filtration system.

It should be emphasized that the present invention is not aimed attreating open angle glaucoma without surgery ba using prostaglandins ofthe A and J type for the intraocular pressure reducing effect of theseprostaglandins, an invention described e.g. in U.S. Pat No. 4,883,819(Bito), but solely for preventing the scar formation that occurs in thefiltration bleb and fistula created by surgery.

EXAMPLES

The invention is exemplified by the following non-limiting examples:

The following prostaglandins, purchased from Cayman Chemicals, AnnArbor, Mich., USA, and Biomol Feinchemikalien GmbH, Hamburg, Germanywere used in the experiments: prostaglandin A₁ (PGA₁), prostaglandin A₂(PGA₂), prostaglandin J₂ (PGJ₂) and 15-deoxy-Δ^(12,14)-prostaglandin J₂(15-Δ^(12,14)-PGJ₂). The chemical structures of the test compounds aredepicted in FIG. 1. All test compounds were stored at −20° C., and freshsolutions containing the appropriate concentrations of the testcompounds were made every second day. The test compounds were initiallydissolved in ethanol and then diluted in the culture medium.

Human fibroblasts were used at passage 22-24. The culture mediumconsisted of DMEM with 10% fetal calf serum and 50 μg/ml gentamycin.Around 10,000 cells were seeded into each well of 48 well microtiterplates. The total volume of culture medium was about 250 μl/well. Thecells were incubated at 37° C. with 5% CO₂ humidified air. The culturemedium was changed every second day, and at the same time new, freshlyprepared prostaglandin was added to the medium. The test compounds wereinvestigated at several concentrations in the range of 10⁻⁶ to 10⁻³Moles/l. Preliminary studies indicated that concentrations lower than10⁻⁶ M had no effect. The concentrations in the figures depicting theresults are expressed as the negative logarithm The culture medium aloneserved as control. After 5-7 days of culture the experiment wasterminated by fixation of the cells in 1% glutaraldehyde. The cells werethen stained with 0.1% crystal violet, and the stain was eluted with2.5% sodium lauryl sulphate. The absorbance of the colored solutions wasdetermined spectrophotometrically. Each experiment was performed atleast in duplicate.

The results of the ex tents are depicted in FIGS. 2-3. It can be seenthat all test compounds, PGA₁, PGA₂, PGJ₂ as well as15-deoxy-Δ^(12,14)-PGJ₂ effectively reduced the cell number of thecultures as shown by the decrease in crystal violet absorbance. Thecontrol wells (without prostaglandin) exhibited absorbances similar tothe lowest concentration tested of the test compounds (not shown in thefigures). Although all test compounds exhibited the same absoluteefficacy in reducing the cell number, PGA₂ appeared to be the mostpotent one, and exhibited an EC₅₀ value of around −5.5 log unitscorresponding to about 3×10⁻⁶ Moles/l. The corresponding value of PGJ₂was about −4.75 log units corresponding to about 2×10⁻⁵ Moles/l, whereasthe values of the two other prostaglandin analogues were clearly higher.

Concentrations in the range of 10⁻⁵ to 10⁻⁴ Moles/l are possible toachieve in the filtration bleb under the conjunctiva in vivo, and sincethese concentrations had marked effect in vitro, it is very likely thateven lower concentrations of the test compounds would exbibit beneficialantiproliferative effect on the fibroblasts in the filtration bleb. Thusprostaglandins with the general structure of cyclopentenone such asPGA's and PGJ's seem to have a marked antiproliferative and indeed evencytotoxic effect depending on concentration and this property of theseprostaglandins can be used clinically to prevent scaring of thefiltration bleb after glaucoma surgery.

Two series of in vivo experiments have been performed in rabbits duringthe priority year. In the first series of experiments it wasdemonstrated that the intraocular pressure is maintained at a reducedlevel after modified trabeculectomy surgery during topical treatmentwith PGA₂-isopropyl ester (PGA₂-IE) eye drops, and in the second seriesof experiments it was shown that in addition to the antiproliferativeeffect, cyclopentenone prostaglandins also have a markedanti-inflammatory effect in the eye which is advantageous after glaucomasurgery.

Maintenance of the Intraocular Pressure at a Reduced Level afterGlaucoma Surgery by Cyclopentenone Prostaglandin Treatment.

Ten New Zealand White adult rabbits underwent modified unilateraltrabeculectomy surgery and were treated with corticosteroids andmydriatics postoperatively for about one month according to a clinicalprotocol. Starting the same day as the surgery was performed, theoperated eye in five animals was in addition treated twice daily withPGA₂-IE eye drops (10 μg/dose) for 4 months, while in the other fiveanimals the operated eye was treated with vehicle only for the sameperiod of time. The intraocular pressure was measured throughout thetreatment period at least every second week. The results are presentedin FIGS. 4 and 5, and demonstrate that the intraocular pressure of theoperated eyes throughout the treatment period in all animals wasmaintained at a lower level than in the contralateral (non-operated)control eye. However, with time there was a clear tendency towardsbetter maintenance of the intraocular pressure at a reduced level aftersurgery in the animals treated with PGA₂-IE than in the vehicle controlanimals. From FIG. 4 it is apparent that the difference in intraocularpressure between the vehicle-treated operated and control eyes steadilydiminished with time which was quite opposite to the difference intraocular pressure between the PGA₂-IE-treated operated and control eyesin FIG. 5. Thus it appears that even during this short treatment period(4 months) a difference in the level of intraocular pressure reductionbetween the PGA₂-IE treated and vehicle-treated operated eyes ismanifested, and at the end of the treatment period the difference inintraocular pressure reduction was statistically significant (p<0.05)(Table 1). It is also evident that the PGA₂-IE treatment had noadversarial effects in the eye as there were no signs of localinflammation or increased intraocular pressure during the treatmentperiod that could be attributed to the use of the prostaglandinanalogue.

TABLE 1 Intraocular pressure at the end of the 4 month treatment periodfollowing surgery. The operated eyes received PGA₂-IE or vehicle, thecontrol eyes received no treatment. Treatment Operated eye Control eyeDifference group n (mmHg) (mmHg) (mmHg) PGA₂-IE 5 17.2 ± 2.9 21.0 ± 2.4 3.8 ± 1.0* Vehicle 5 19.8 ± 1.5 20.8 ± 1.6 1.0 ± 0.3 *p < 0.05 whencompared to vehicle group

Anti-inflammatory Effect of Cyclopentenone Prostaglandins in the Eye

Ten New Zealand White adult rabbits underwent extracapsular lensextraction in one eye to investigate the effect of PGA₂-E onpostsurgical inflammation. Lens extraction was used as a model sincethis procedure is always followed by some inflammatory changes. Thecontralateral eye was used as an untreated control eye in each animal.The operated eyes were given routine postsurgical treatment withcorticosteroids and mydriatics for about one month. From the day afterthe surgery and for a total of 4 months 5 of the animals were treatedwith PGA₂-IE eye drops twice daily (10 μg/dose) in the operated eyewhile the contralateral control (non-operated) eye received notreatment. The other 5 animals were treated with the vehicle only twicedaily for the same period of time. Surprisingly it was found that onlyone of the animals receiving PGA₂-IE exhibited signs of clear-cutinflammation whereas most of the animals in the vehicle group repeatedlyexhibited signs of intraocular inflammation during the 4 month treatmentperiod. The signs of inflammation comprised miosis, iridial hyperemia,flare and fibrin accumulation in the anterior chamber, and additionalanti-inflammatory medication had to be given to these animals. ThusPGA₂-IE had a remarkable anti-inflammatory effect in the eye. Theresults are summarized in Table 2.

TABLE 2 Number of animals exhibiting inflammation of the operated eye 2and 4 months after surgery. Time of treatment after surgery Treatment 2months 4 months group Normal Inflamed Normal Inflamed Vehicle 0 5 1 4PGA₂-IE 3  2* 4 1 *one only slightly inflamed

Since inflammation, which also occurs in the filtering bleb aftertrabeculectomy surgery, is known to induce fibroblast proliferation andscarring, reduced inflammation is beneficial for keeping the filteringbleb functional.

Accordingly, prostaglandins of the subtypes A and J may be utilised toprevent scar formation of the filtration bleb and drainage fistula afterglaucoma surgery. In the exemplification the present inventors have used4 different cyclopentenone prostaglandins, but also other analogues ofthe A and J cyclopentenone prostaglandins may be employed. Suchanalogues comprise e.g. 16,16-dimethyl-PGA₁, Δ⁷-PGA₁, and16,16-dimethyl-PGA₂ as well as PGJ₁, Δ¹²PGJ₁ and Δ¹²PGJ₂. There are alsoother types of derivatives of prostaglandin A and J which are known fromthe literature and which are obvious candidates to be used according tothe present invention. One such group constitutes derivatives witharomatic or non-aromatic ring substituted omega chain as disclosed inPCT application SE89/00475. Also alpha chain modified prostaglandins ofthe A and J type may be employed. The alpha chain may be straight orbranched, saturated or unsaturated with or without carboxylic acid,ester, amide, alcohole or ether moieties. The chain may also contain anaromatic or non-aromatic ring.

In addition to the above, the present inventors have recently found thatprostalandins carrying a hydroxyl substituent on carbon 18-20 exhibit noor little initative effect. Such modifications of cyclopentenoneprostaglandins may also be employed on the substances according to thepresent invention.

PGA and PGJ or their analogues may be modified to more lipophilicsubstances and/or more stable substances e.g. by converting the acidmoiety to alcohole, ether, amide or ester. Such esters that may beemployed clinically comprise e.g. alkyl esters with 1-10 carbon atoms,and especially short alkyl esters e.g. methyl, ethyl, isopropyl andcyclic esters such as benzyl.

The prostaglandin compounds and their esters or derivatives should beused in a suitable ophthalmologically compatible vehicle. Such vehiclesinclude as described previously aqueous and oil solutions as well ascreams and ointments. The vehicle may contain solubilizers andstabilizers such as cyclodextrins, micelle systems, nanoparticles,polymeres and various slow-release systems. The vehicles may or may notcontain preservatives such as benzalkonium chloride, chlorhexidine,thiomersal, parabenzoic acid, and other compounds with satisfactoryantimicrobial activity.

Accordingly, the prostaglandin of A or J type should be used topicallyon the eye for different period of time to prevent scar formation in thefiltration bleb and drainage fistula after glaucoma surgery. Suchtreatment may be continuous or intermittent, and it may be necessaryalso to use the medication only for a relatively short period of timeimmediately after surgery.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionas set forth in the claims appended hereto.

What is claimed is:
 1. A method for the prevention of scar formation inthe filtration bleb and drainage fistula after glaucoma surgeryperformed on a human eye, characterized in that a composition containinga prostaglandin of subtype A or J is brought in contact with the surfaceof the eye.
 2. The method according to claim 1, characterized in thatthe prostaglandin is PGA or a derivative thereof.
 3. The methodaccording to claim 2, characterized in that the prostaglandin is PGA₂ ora derivative thereof.
 4. The method according to claim 3, characterizedin that the prostaglandin is 17-phenyl-18, 19, 20-trinor-PGA₂.
 5. Themethod according to claim 1, characterized in that the prostaglandin isPGJ or a derivative thereof.
 6. The method according to claim 5,characterized in that the prostaglandin is PGJ₂ or a derivative thereof.7. The method according to claim 6, characterized in that theprostaglandin is 17-phenyl-18,19,20-trinor-PGJ₂.
 8. The method accordingto any one of claims 1 through 7, characterized in that theprostaglandin is an ester, especially an isopropyl ester of theprostaglandin in question.
 9. The method according to any one of claims1 through 7 characterized in that the prostaglandin is administeredtopically to the eye in an amount in the interval of 0.1 to 500 μg ofthe active substance per application.
 10. The method according to anyone of claims 1 though 7 characterized in that the prostaglandincomposition is administered topically to the eye 1 to 3 times daily,once every second day, once a week, or intermittently, duringadministration periods followed by periods when no administration isperformed.
 11. The method according to any one of claims 1 through 7characterized in that the prostaglandin is administered topically to theeye with the aid of a slow release drug insert.
 12. The method accordingto any one of claims 1 though 7 wherein the prostaglandin isadministered topically to the eye in an amount of 1-30 μg perapplication.